Electrochemical assay of plasminogen activators in plasma using polyion-sensitive membrane electrode detection

Two relatively simple electrochemical assay methods suitable for the measurement of plasminogen activators (including urokinase (u-PA), streptokinase (SK), and tissue plasminogen activator (t-PA)) in plasma samples are described. In one approach, the initial rate of decrease in the potentiometric response of a polycation-sensitive membrane electrode toward protamine is monitored after addition of a preincubated reaction mixture containing the sample and exogenous plasminogen. The plasmin formed from plasminogen by the activators catalyzes the decomposition of the arginine-rich protamine substrate, yielding smaller polycationic fragments that are not sensed by the electrode. Alternately, the sample, plasminogen, and protamine can be incubated together, and the remaining protamine in this reaction mixture can be measured at a fixed point in time by placing the electrode into the mixture and recording the electromotive force response. Working curves found with both methods for plasma samples spiked with varying levels of the activators cover the expected therapeutic activity ranges found in the plasma of patients treated with these "clot-busting" drugs.     

Paclitaxel quantification in mouse plasma and tissues containing liposome-entrapped paclitaxel by liquid chromatography-tandem mass spectrometry: application to a pharmacokinetics study

A liquid chromatography-tandem mass spectrometry assay to quantify total paclitaxel in mouse plasma and tissue homogenates containing paclitaxel, Taxol, or liposome-entrapped paclitaxel-easy to use (LEP-ETU) was developed and validated. Docetaxel was used as the internal standard (IS). Liquid-liquid extraction with tert-butyl methyl ether was used for plasma sample preparation, and a one-step protein precipitation with acetonitrile containing 0.1% acetic acid was developed for tissue homogenates. Paclitaxel and IS are separated on a 50 x 2.1-mm C18 column and quantified using a triple-quadrupole mass spectrometer operating in positive ion electrospray multiple reaction monitoring mode, with a total run time of 3.5 min. The peak area of the m/z 854.4--> 286.2 transition of paclitaxel is measured versus that of the m/z 808.5--> 527.5 transition of IS to generate the standard curve. In plasma, the linear range is 0.2-500 ng/mL and could be extended by dilution to 100,000 ng/mL with acceptable precision and accuracy (< or = 15%). The lower limit of quantification is 0.5 ng/mL in tissue homogenates (10 ng/g tissue), and the standard curve is linear up to 1000 ng/mL, with precision and accuracy < or = 15%. This assay was used to support a pharmacokinetics and tissue distribution study of LEP-ETU in mice.     

Effect of fasting on the clearing factor lipase (lipoprotein lipase) activity of fresh and defatted preparations of rat heart muscle

The clearing factor lipase (lipoprotein lipase) activities of homogenates of fresh tissue and of acetone-ether powders have been compared in hearts from fed and starved rats. The activity of the enzyme measured in homogenates of acetone-ether powders is generally higher than that in homogenates of the fresh tissue. Activation is due to an effect of the acetone-ether treatment on enzyme which is associated with the tissue residue in fresh tissue homogenates. A similar activation occurs when the tissue residue is treated with deoxycholate. When rats are fasted, a marked increase in the clearing factor lipase activity of the heart occurs. Peak activities are reached after 10-24 hr, and thereafter the activity falls slowly. This pattern of activity is observed in homogenates of fresh tissue and of acetone-ether powders. The activity of clearing factor lipase in diaphragm muscle also increases in rats starved for 8 or 24 hr. The importance of the change in muscle clearing factor lipase activity on fasting in relation to triglyceride fatty acid utilization by this tissue is emphasized.     

The histochemistry of isocitric and oestradiol-17 beta dehydrogenases in the endometrium of postmenopausal women treated with oestrogens and progestogens

Summary Endometrium was obtained from postmenopausal women during treatment with either oestrogen alone or on the third, sixth, tenth or twelfth day of combined therapy with oestrogens and progestogens. The activities of oestradiol-17 and isocitric dehydrogenases were measured in homogenates of the whole tissue and the enzymes were also located histochemically. Oestradiol dehydrogenase was located exclusively within the epithelium, whilst isocitric dehydrogenase was found in both epithelium and stroma, and also in stromal arterioles. Histochemically, oestradiol dehydrogenase was found in all the specimens which exhibited biochemical activity but in none of those from which it was absent. Isocitric dehydrogenase however, was seen in all tissue sections despite widely varying levels of biochemical activity in the homogenate. The method for measuring isocitric dehydrogenase activity was therefore nonspecific, whilst that for oestradiol dehydrogenase was reliable and low levels of enzyme activity could be detected. The latter technique may therefore be useful to predict the sensitivity of endometrial carcinomata to progestogens.     

Characterization of succinate dehydrogenase and alpha-glycerophosphate dehydrogenase in pancreatic islets

Succinate dehydrogenase activities in homogenates of rat and ob/ob mouse pancreatic islets were only 13% of the activities in homogenates of liver and were also several times lower than in homogenates of pancreatic acinar tissue. This indicates that the content of mitochondria in pancreatic islet cells is very low. The very low activity of succinate dehydrogenase is in agreement with the low mitochondrial volume in the cytoplasmic ground substance of pancreatic islet cells as observed in morphometric studies. This may represent the poor equipment of pancreatic islet cells with electron transport chains and thus provide a regulatory role for the generation of reducing equivalents and chemical energy for the regulation of insulin secretion. The activities of succinate dehydrogenase in tissue homogenates of pancreatic islets, pancreatic acinar tissue, and liver were significantly inhibited by malonate and diazoxide but not by glucose, mannoheptulose, streptozotocin, or verapamil. Tolbutamide inhibited only pancreatic islet succinate dehydrogenase significantly, providing evidence for a different behavior of pancreatic islet cell mitochondria. Therefore diazoxide and tolbutamide may affect pancreatic islet function through their effects on succinate dehydrogenase activity. The activities of alpha-glycerophosphate dehydrogenase in homogenates of pancreatic islets and liver from rats and ob/ob mice were in the same range, while activities in homogenates of pancreatic acinar tissue were lower. None of the test agents affected alpha-glycerophosphate dehydrogenase activity. Thus the results provide no support for the recent contention that alpha-glycerophosphate dehydrogenase activity may be critical for the regulation of insulin secretion.     

Inhibition of carbonic anhydrase by plasma of dogs and rabbits

Carbonic anhydrase (CA) activity was measured by the Bowes-Davis technique in diluted hemolysates of dog erythrocytes, rabbit erythrocytes, and dog lung tissue homogenates. Plasma (from the same animal) inhibited the CA activity in each case. For 1:16,700 dilution of dog erythrocytes, the CA catalyzed the CO2 hydration reaction by 5.3 +/- 0.4-fold above the uncatalyzed rate, and half that activity was inhibited by plasma concentrations of 0.44 +/- 0.05%. Similar rabbit CA concentrations were inhibited by plasma concentrations of 1.02 +/- 0.24%. CA from dog lung tissue homogenate is only partially inhibited by plasma even at high plasma concentrations, suggesting different isozymes, at least one of which is not inhibited by plasma. The results suggest that extrapolating from artificially perfused lungs or histological observations to in vivo conditions may not be valid, and the possibility of inhibition by plasma in at least some species should be considered.     

Estimation of Steroids and Steroid Metabolism in a Lower Vertebrate (Lampetra planeri Bloch)

Pregnenolone, androstenedione and testosterone were identified by RIA in tissue homogenates of the pronephric region, the opisthonephros, the gonads and in plasma samples from male and female immature and mature adult brook lampreys. Additionally, hydroxysteroid dehydrogenase activity was determined spectrophotometrically in homogenates from the same tissues of mature and spent adult brook lampreys employing pregnenolone, testosterone or 3β,17β-dihydroxy-5β-androstane as substrates. The steroid levels show differences corresponding to developmental stages, tissues and sex. Remarkable quantities of testosterone were measured in the testicular tissue homogenates, in homogenates obtained from the pronephric region and in the serum.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

Diminished activities of fatty acid synthesis enzymes in insulin-resistant adipocytes from spontaneously obese rats.

Acetyl coenzyme A carboxylase and fatty acid synthetase activities were studied to determine the biochemical basis of the markedly impaired capacity of fat cells from spontaneously obese, old rats to convert glucose to fatty acids relative to cells from lean, young rats. Michaelis constants for the substrates of both enzymes were similar in large and small adipocyte homogenates. In contrast, Vmax values were over 80% less in homogenates from large relative to small cells on a per cell basis. Long-term dialysis or the presence of albumin during the assays failed to restore the activities of these enzymes in homogenates of large fat cells. The combination of equal volumes of homogenates from the two cell types resulted in carboxylase and synthetase activities intermediate between activities found in the two homogenates alone. Therefore, the presence of endogenous allosteric inhibitors does not appear to account for the markedly blunted fatty acid synthesis enzyme activities in large fat cells. These results suggest that the fatty acid synthesis impairment, which is a primary defect in the insulin resistance of the large cells, is at least partly due to diminished cellular contents of acetyl coenzyme A carboxylase and fatty acid synthetase.     

Structural equivalents of latency for lysosome hydrolases.

1. Structure-linked latency, a trait for most lysosome hydrolase activities, is customarily ascribed to the permeability-barrier function performed by the particle-limiting membrane, which shields enzyme sites from externally added substrates. 2. The influence of various substrate concentrations on the reaction rate has been measured for both free (non-latent) and total (completely unmasked by Triton X-100) hydrolase activities in rat liver cell-free preparations. The substrates were: beta-glycerophosphate, phenolphthalein mono-beta-glucuronide. p-nitrophenyl N-acetyl-beta-D-glucosaminide and p-nitrophenyl beta-D-galactopyranoside. The ratio (free activity/total activity) X 100 is called fractional free activity at any given substrate concentration. 3. The fractional free activity of beta-glucuronidase and beta-N-acetylglucosaminidase were clearly independent of substrate concentration, over the range examined, in both homogenates and lysosome-rich fractions. The fractional free activity of acid phosphatase appeared to be either unaffected (homogenate) or even depressed (lysosome-rich fraction) by increasing the beta-glycerophosphate concentration. The fractional free activity of beta-galactosidase consistently showed a non-linear increase with increasing substrate concentration in both homogenates and lysosome-rich fractions. 4. Procedures such as treatment with digitonin, hypo-osmotic shock and acid autolysis, although effective in causing varying degrees of resolution of the latency of lysosome hydrolase activities, were unable to modify appreciably the pattern of dependence or independence of their fractional free activities on substrate concentration, as compared with that exhibited by control preparations. Ouabain did not affect the free beta-N-acetylglucosaminidase activity of liver homogenates at all. 5. Preincubation of control preparations with beta-glycerophosphate or p-nitrophenyl beta-galactoside did not result in any significant stimulation of the free hydrolytic activity toward these substrates. 6. The results consistently support the view that the membrane of "intact" lysosomes is virtually impermeable to all the substrates tested, except for p-nitrophenyl beta-galactoside, for which the evidence is contradictory. Moreover the progressive unmasking of the hydrolase activities produced by these procedures in vitro reflects the increasing proportion of enzyme sites that are fully accessible to their substrates rather than a graded increase in the permeability of the lysosomal membrane.     

Rat renal gamma-glutamyltransferase activity: a reaction of glutamine synthetase

The experimental observations of Welbourne (Am. J. Physiol. 226, 544-548 (1974)) led him to propose that a gamma-glutamyltransferase activity contributes significantly to basal renal ammonia production. He measured the transferase activity in vitro as gamma-glutamylhydroxamate or ammonia production from glutamine and hydroxylamine. We report that in crude homogenates of rat kidney, gamma-glutamyltransferase activity requires the presence of a divalent cation, arsenate, and ADP. These conditions are also required for the maximal expression of the gamma-glutamyltransferase reaction catalyzed by glutamine synthetase. Glutamine synthetase and gamma-glutamyltransferase activities exhibit an identical distribution during differential centrifugation. In addition, the two activities comigrate sucrose gradient velocity centrifugation, and exhibit identical heat inactivation and L-methionine sulfoximine inhibition profiles. Furthermore, both activities are found to be localized primarily in the outer stripe region of the renal medulla. Based on these observations, it is concluded that the primary gamma-glutamyltransferase activity as assayed in rat renal tissue is a partial reaction of glutamine synthetase, an enzyme which does not catalyze the production of ammonia and gamma-glutamyl peptides from glutamine.     

The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339.

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.     

REGIONAL DISTRIBUTION AND PROPERTIES OF THE GLYCINE CLEAVAGE SYSTEM WITHIN THE CENTRAL NERVOUS SYSTEM OF THE RAT: EVIDENCE FOR AN ENDOGENOUS INHIBITOR DURING IN VITRO ASSAY

—The activity of the glycine cleavage system (GCS) was determined in homogenates from five specific regions of the rat CNS (telencephalon, midbrain, cerebellum, medulla-pons, and spinal cord). An inverse trend was noted between the glycine content and the specific activity of the GCS in the regions. A 25-fold range in the enzyme activities was found between the telencephalon (highest) and the spinal cord (lowest). The properties of the GCS activity in CNS homogenates agreed with those properties previously described for this system in partially purified preparations of liver and brain mitochondria (Kikuchi , 1973; Bruin et al., 1973). Within the CNS homogenates, the liberation of CO₂ from the carboxyl carbon of glycine was quantitatively coupled to the formation of serine. The presence of an endogenous inhibitor(s) within neural tissues was suggested by the non-additivity of the activities when homogenates from the various regions were combined. Moreover, homogenates of CNS tissue inhibited the GCS activity of liver homogenates, and an inverse relationship was found between the level of GCS activity in a given region of the CNS and its ability to inhibit the GCS activity of liver homogenates. This inhibition of liver activity was greatest when liver was incubated with homogenates of spinal cord (86%) and lowest when incubated with homogenates of telencephalon (20%). Because of this endogenous inhibition, the apparent activity of the GCS measured in vitro may not reflect the contribution of this enzyme system in the metabolism of glycine in vivo. Although the significance of this inhibition is not known, a possible role is discussed for the regulation of the levels in glycine and one-carbon pools within the CNS.     

Age-dependent lipid peroxidation in neonatal rat lung tissue.

A unique, age-specific pulmonary lipid peroxidation has been found to occur after incubation of neonatal rat lung homogenates in the absence of any added factors. As measured by the formation of malondialdehyde, lipid peroxidation was not detectable in rat lung homogenates prepared from animals immediately after birth but appeared by the second day and reached a maximum at 5 days of age. The effect gradually disappeared by 20 to 21 days after birth. The addition of NADPH did not enhance lipid peroxidation in the sensitive age group nor did it initiate lipid peroxidation when added to lung homogenates from either 1-day-old or adult rats. The activities and concentrations of various endogenous antioxidants were measured in neonatal lung tissue. When measured in lung tissue obtained from rats during the sensitive age period, no concomitant deficiencies of glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase, reduced glutathione, or a-tocopherol were observed. With the exception of α-tocopherol, none of these factors inhibited malondialdehyde formation when added to homogenized lung tissue prepared from 5-day-old rats. α-Tocopherol did inhibit malondialdehyde formation in 5-day-old rat lung homogenates but at a concentration much greater than the endogenous concentration found in adult rat lungs. The 21-day neonatal age period during which malondialdehyde is produced following incubation of lung tissue is similar to the 3-week period immediately after birth reported to be the time of maximum proliferation of rat lung fibroblasts, type 1 pneumocytes and type II pneumocytes.     

Catalase activity measured with a micro oxygen electrode in a pressurized reaction vessel

The assembly and the use of a simple airtight pressurized reaction vessel are described for the measurement of catalase activity with a micro oxygen electrode in an optically heterogenous medium. The oxygen concentration is expressed as the ratio of observed current to the current in an air-saturated solution. Thus, an individual standard can be obtained for each measurement and the calibration is less affected by changes in the amplification factor. Different procedures for calibration of the oxygen electrode were compared. Specific activities of crystalline catalase, of red blood cells from humans and from normal or acatalasemic mice, and of liver homogenates from normal or amino-triazole-pretreated rats were determined. The specific catalase activity of human erythrocytes, as found by this method, agrees with that obtained photometrically.     

Localization of adenylate cyclase and 5′-nucleotidase activities in human thyroid follicular cells

Summary The localization of adenylate cyclase and 5-nucleotidase activities in the follicular cells of adenomatous goiter and normal thyroid was studied by light and electron microscopy. Simultanous biochemical measurement for both activities was carried out to confirm the histochemical findings. Adenylyl-imidodiphosphate (AMP-PNP) was used as an effective substrate for adenylate cyclase. The specificity of the adenylate cyclase reaction was also examined by adding oxalacetic acid or PCMB as an adenylate cyclase inhibitor, and by adding sodium fluoride or TSH as an adenylate cyclase stimulator to the reaction mixture. In the case of tissue from adenomatous goiter, a large amount of the reaction product of the adenylate cyclase activity was found uniformly in the apical and lateral plasma membrane and not in the basal plasma membrane. In the cases of normal thyroid, a small amount of the reaction product of adenylate cyclase activity was demonstrated, and only in the lateral plasma membrane of the follicular cells. On the other hand, the histochemical localization of 5-nucleotidase activity was the same in adenomatous goiter and normal thyroid. The reaction product of 5-nucleotidase activity was found predominantly in the apical plasma membrane of the follicular cells. The biochemical findings indicated that the activity of adenylate cyclase per gram tissue was approximately 2 times higher in the case of adenomatous goiter than that in the case of normal thyroid, while the 5-nucleotidase activity in adenomatous goiter was in slightly higher level than in normal thyroid. Thus the histochemically demonstrable amount of adenylate cyclase and 5-nucleotidase reflected the activity levels measured biochemically. The lack of demonstrable adenylate cyclase activity in the basal plasma membrane suggests the possibility that this structure may not play any important role in TSH reception.     

Oxidative stress in streptozotocin-induced diabetic rats: effects of garlic oil and melatonin

In the present study, oxidative stress in diabetic model and the effect of garlic oil or melatonin treatment were examined. Streptozotocin (60 mg/kg body weight, i.p.)-induced diabetic rats, showed a significant increase of plasma glucose, total lipids, triglyceride, cholesterol, lipid peroxides, nitric oxide and uric acid. Concomitantly, significant decreases in the levels of antioxidants ceruloplasmin, albumin and total thiols were found in the plasma of diabetic rats. Lipid peroxide levels were significantly increased in erythrocyte lysate and in homogenates of liver and kidney, while superoxide dismutase (SOD) activities were decreased in tissue homogenates of liver and kidney. Treatment of diabetic rats with garlic oil (10 mg/kg i.p.) or melatonin (200 microg/kg i.p.) for 15 days significantly increased plasma levels of total thiol, ceruloplasmin activities, albumin. Lipid peroxides, uric acid, blood glucose, total lipid, triglyceride and cholesterol were decreased significantly after treatment with garlic oil or melatonin. Nitric oxide levels were decreased significantly in rats treated with melatonin only. In erythrocytes lysate, glutathione S-transferase (GST) activities were increased significantly in rats treated with garlic oil or melatonin, while lipid peroxides decreased significantly and total thiol increased significantly in melatonin or garlic oil treatment, respectively. In liver homogenates of rats treated with garlic or melatonin, lipid peroxides were decreased significantly, and GST activities increased significantly, while SOD activities were increased significantly in liver and kidney after garlic or melatonin treatment. The results suggest that garlic oil or melatonin may effectively normalize the impaired antioxidants status in streptozotocin induced-diabetes. The effects of these antioxidants of both agents may be useful in delaying the complicated effects of diabetes as retinopathy, nephropathy and neuropathy due to imbalance between free radicals and antioxidant systems. Moreover, melatonin may be more powerful free radical scavenger than garlic oil.     

Harvesting and separation of two populations of lysosomes from porcine endometrium

The lysosomes present in homogenates of porcine endometrium epithelium equilibrate in two density regions of Percoll gradients. Patterns with varying proportions between high and low density peaks are observed, when aliquots of a tissue sample are processed with different all-glass Potter-Elvejhem homogenizers. The described constant-tolerance shearing device (CTSD), in contrast, provides homogenate fractions with higher latencies and steady distribution patterns. They are characteristic for each of the six lysosomal markers and the six other structure-bound enzymes measured in gradient fractions of the particulate matter harvested between 600g and 17,000g. The 17,000g sediments of CTSD homogenates contain more than 40% of the total lysosomal enzymatic activities. Recoveries from Percoll gradients are between 93 and 101%. Enrichments in the high density region range from 35-fold (beta-glucosidase) to 82-fold (acid ribonuclease). Both lysosomal populations exhibit latencies between 89 and 94%. Our results indicate that light lysosomes can be artificially generated by inappropriate homogenization, which should be considered in experiments on the formation and maturation of lysosomes.     

Metabolism of free and membrane-bound ribosomes during aging of Jerusalem artichoke tuber slices

Summary A biochemical and cytochemical study has been made of the distribution of -glycerophosphatase (EC 3.1.3.2) activity in mature and differentiating phloem cells of Nicotiana tabacum L. and the pH dependence and kinetics of -glycerophosphate hydrolysis of homogenates of fresh leaf midveins and midveins fixed in formaldehyde-gluteraldehyde. -glycerophosphatase showed two peaks of activity at pH 5.5 and 6.2. Enzyme saturation kinetics were exhibited by both fresh and fixed tissue homogenates. At a substrate concentration of 2 mM, 65% of the enzyme activity survived fixation. Specimens for cytochemical localization were incubated with 2 mM -glycerophosphate at pH 5.5 and 6.2. Specimens showed consistent patterns of reaction product deposition. Little or no reaction product was deposited in controls incubated without substrate or with substrate plus 0.01 M fluoride. -glycerophosphatase activity in the phloem and xylem is considerably higher than in surrounding tissue. Dense localization of reaction product was demonstrated on the vacuolar membranes, the inner membranes of mitochondria, and the dictysomes of phloem parenchyma and companion cells. The plasma membrane and endoplasmic reticulum cisternae of these cells were usually free of reaction products. Enzyme activity in mature sieve elements was associated with the parietal and stacked systems of endoplasmic reticulum and with the P-protein. There was inconsistency of staining of P-protein in mature sieve elements although the association of reaction products with the P-protein appeared to show a correlation with maturity and dispersal. The P-protein bodies of differentiating sieve elements showed no reaction product deposition. The distribution of -glycerophosphatase activity has been compared with that previously recorded for ATPase activity in the phloem of Nicotiana tabacum.